Adipokines and Vascular Endothelial Growth Factor in Normal Human Breast Tissue in Vivo - Correlations and Attenuation by Dietary Flaxseed

Abstract

Exposure to sex steroids increases the risk of breast cancer but the exact mechanisms are yet to be elucidated. Events in the microenvironment are important for carcinogenesis. Diet containing phytoestrogens can affect the breast microenvironment and alter the risk of breast cancer. It has previously been shown that estrogen regulates extracellular levels of leptin, adiponectin, and VEGF in normal breast tissue in vivo. Whether these proteins correlate in breast tissue in vivo or if diet addition of flaxseed, a major source of phytoestrogens in Western diets, alters adipokine levels in breast tissue are unknown. We used microdialysis to sample proteins of normal human breast tissue and abdominal subcutaneous fat in situ in 34 pre-and postmenopausal women. In vitro, co-culture of breast cancer cells and primary human adipocytes was used. In vivo, in normal breast tissue, a significant positive correlation between VEGF and leptin was detected. No correlations were found in fat tissue. Co-culture of adipocytes and breast cancer cells per se increased the secretion of VEGF and leptin and enhanced the effects of estradiol compared to culture of either cell type alone. In vitro, inhibition of VEGF diminished the release of leptin while inhibition of leptin had no influence on VEGF secretion. The levels of leptin decreased and adiponectin increased after a dietary addition of 25 g of flaxseed/day for one menstrual cycle. We conclude that VEGF and leptin correlate significantly in normal human breast tissue in vivo and that dietary addition of flaxseed affect adipokine levels in the breast.

Keywords: Adiponectin; Diet; Estrogen; Flaxseed; Leptin; Microdialysis.

Fig. 1

VEGF correlated with leptin and the ratio of leptin:adiponectin in normal human breast tissue. Microdialysis was used for sampling of extracellular VEGF, leptin, and adiponectin in vivo in normal breast tissue (extracellular breast) and the abdominal subcutaneous (s.c.) fat (extracellular fat), in pre-and postmenopausal women. Filled circles represent women investigated in the luteal phase of the menstrual cycle and open circles women investigated in the follicular phase of the menstrual cycle. Open squares represent postmenopausal women. a. Extracellular VEGF levels correlated positively with extracellular leptin levels in normal breast tissue, r = 0.6; p < 0.0001; n = 43, but not in the abdominal s.c. fat, r = 0.22; p = 0.15; n = 41. b. VEGF did not correlate with adiponectin in normal breast tissue, r = 0.15; p = 0.32; n = 43, or abdominal s.c. fat tissue, r = 0.16; p = 0.31; n = 41. c. There was a significant positive correlation between VEGF and the ratio of leptin to adiponectin in normal breast tissue, r = 0.4; p < 0.01; n = 43, whereas no correlation was found in abdominal s.c. fat tissue, r = 0.23; p = 0.14; n = 41

Fig. 2

Inhibition of VEGF decreased leptin secretion in co-culture of MCF7 and adipocytes. Co-culture of ER+ breast cancer cells, MCF7, and freshly isolated human adipocytes, were exposed to; a. Anti-VEGF antibody at 0.1 μg/ml and 1 μg/ml. Extracellular levels of leptin were analyzed after 48 h. *, p < 0.05 and ****, p < 0.0001 compared with controls, n = 5–6 in each group. b. Anti-leptin antibody at 0.1 μg/ml and 1 μg/ml. Extracellular levels of VEGF were analyzed after 48 h. n = 5–6 in each group. c. Anti-VEGF antibody at 0.1 μg/ml and 1 μg/ml in the presence of estradiol (10⁻⁹ M). Extracellular levels of leptin were analyzed after 48 h. ***, p < 0.001 and ****, p < 0.0001 compared with controls, n = 5–6 in each group. d. Anti-leptin antibody at 0.1 μg/ml and 1 μg/ml in presence of estradiol (10⁻⁹ M). Extracellular levels of VEGF were analyzed after 48 h. n = 5–6 in each group

Fig. 3

Co-culture with adipocytes enhanced the effects of estradiol on leptin and VEGF secretion. A. MCF7 cells, freshly isolated human adipocytes or co-culture of MCF7 and adipocytes, were cultured with or without estradiol (10⁻⁹ M) for 48 h. Extracellular levels of VEGF were measured in the conditioned medium with ELISA. ***, p < 0.001 and ****, p < 0.0001, n = 6. B. MCF7 cells alone or co-cultured with adipocytes were treated with estradiol (10⁻⁹ M) or estradiol (10⁻⁹ M) and fulvestrand (10⁻⁶ M) for 48 h. Extracellular levels of VEGF were measured with ELISA in the conditioned medium. ***, p < 0.001 and ****, p < 0.0001, n = 6. C. MCF7 cells, adipocytes or co-culture of MCF7 and adipocytes, were cultured with or without estradiol (10⁻⁹ M) for 48 h. Extracellular levels of leptin were measured with ELISA in the conditioned medium. ***, p < 0.001, n = 6. D. MCF7 cells alone or co-cultured with adipocytes were treated with estradiol (10⁻⁹ M) or estradiol (10⁻⁹ M) and fulvestrant (10⁻⁶ M) for 48 h. Extracellular levels of leptin were measured with ELISA in the conditioned medium. *, p < 0.05 and ***, p < 0.001, n = 6. E. MCF7 cells, adipocytes or co-culture of MCF7 and adipocytes, were cultured with or without estradiol (10⁻⁹ M) for 48 h. Extracellular levels of adiponctin were measured with ELISA in the conditioned medium. n = 6 in each group

Fig. 4

Dietary addition of flaxseed decreased leptin and leptin:adiponectin ratio in normal human breast tissue in vivo. Microdialysis of normal human breast tissue and abdominal subcutaneous (s.c.) fat was performed in two consecutive luteal phases in 10 premenopausal women before and after the addition of 25 g of ground flaxseed/day to their diet. *p < 0.05, **p < 0.01, n = 10